摘要
目的通过检测茄病镰刀菌刺激小鼠角膜基质细胞后Toll样受体4(TLR4)的表达分布,探讨TLR4在角膜真菌感染中的作用。方法体外培养小鼠角膜基质细胞,采用茄病镰刀菌液(孢子密度为1×106CFU/mL)来刺激传3代的小鼠角膜基质细胞,于刺激0 h(即未刺激)以及3、6、12 h后应用免疫细胞化学染色、RT-PCR法测定各组细胞TLR4蛋白及mRNA的表达,ELISA法测定各组细胞上清液中肿瘤坏死因子α(TNF-α)的分泌水平。结果培养的角膜基质细胞1周后融合,波形蛋白荧光染色阳性。TLR4蛋白及mRNA在未刺激角膜基质细胞呈微弱表达,3 h较前明显升高,6 h达到高峰,12 h开始减弱,但与0 h相比,差异均有统计学意义(蛋白:t3 h=0.031,t6 h=0.097,t12 h=0.069,P<0.05;mRNA:t3 h=0.367,t6 h=0.422,t12 h=0.078,P<0.05)。TNF-α分泌量随着刺激时间的延长呈上升趋势,6 h达到高峰,然后逐渐下降,3、6、12 h组与0 h组比较差异均有统计学意义(t3 h=21.152,t6 h=40.854,t12 h=27.713,P<0.05)。TLR4mRNA及蛋白的表达与TNF-α的分泌间均呈正相关(r=0.729,0.751,P<0.05)。结论TLR4可能在角膜识别真菌感染、介导炎性防御反应过程中起到重要的作用。
Objective Fungal keratitis is one of the most important factors of blinding eye disease. Present study was to detect the expression of Toll like receptor 4 （TLR4） in cultured mouse keratocytes in vitro stimulated by Fusarium solani suspension and investigate its role to fungal infection of cornea. Methods Forty health Balb/c mice were used in this study following the Standard of Association for Research in Vision and Ophthalmology. The keratocytes of mice were digested using eollagenase Ⅲ and trypsinase and cultured in vitro in DMEM/F12 containing 10% fetal bovine serum. The third generation of ceils were stimulated with Fusarium solani suspension （Spore density was 1 ×10^6 CFU/mL）. Expression of TLR4 protein and mRNA in cultured mouse keratocytes were detected using immunohistochemistry and reverse transcription polymerase chain reaction （ RTPCR） at 0,3,6,12 hours after stimulation. TNF-α in the cells supernatant was detected by ELISA at above-mentioned time points. Results Cultured cells fused and showed the vortex arrangement in the first week after incubation. The cells presented the red fluorescence for vimentin. The immunohistoehemistry and RT-PCR technique determined that expression of TLR4 on cultured keratocytes was weak before stimulation and was gradually stronger after stimulation of Fusarium solani with the strongest expression in 6 hours. The result showed that expression level of TLR4 protein and mRNA was significantly increased at 3 hours, peaked at 6 hours and declined at 12 hours, showing a statistically significant difference between 3,6,12 and 0 hour（ protein：t3h = 0. 031, t6h = 0. 097, t12h = 0. 069, P 〈 0.05 ; mRNA ： t3h = 0. 367, t6h = 0. 422, t12h = 0. 078 , P 〈 0.05 ）. Secrection of TNF-α followed the same pattern to TLR4 （ t3h = 21. 152, t6h = 40. 854, t12h = 27. 713, P 〈 0.05 ）. The positive correlation was found between expression of TLR4 mRNA and protein with IFN-γ secreetion （ r = 0. 729, r = 0.751, P 〈 0. 05 ）. Conclusion TLR4 may play a critical role in recognizing fungi and mediating inflammatory response of cornea.
出处
《眼科研究》
CAS
CSCD
北大核心
2009年第9期747-750,共4页
Chinese Ophthalmic Research
基金
福建省科技厅青年人才项目(2008F3041)
福建医科大学教授发展基金项目(JS06033)资助
